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ABSTRACT: Analysis of 2 cultured normal lung cell lines, Normal Human Bronchial Epithelial (NHBE) and Human Small Airway Epithelial (SAEC) cells (Lonza, Walkersville, MD), following treatment with 5-aza-dC to induce DNA demethylation. These results provide insight into the role of epigenetic alterations, specifically demethylation, in differential gene expression in various lung neoplasms. Overall design: Two normal lung cell lines, NHBE and SAEC, were treated with 5uM 5-aza deoxycytidine for 72 hours and Trichostatin A for 24 hours prior to harvesting total RNA for expression array analysis using the Affymetrix Human Genome U133 Plus 2.0 expression platform. Signal intensity and statistical significance was established for each transcript using dChip version 2005. Two-fold increase based on the 90% confidence interval of the result and expression minus baseline >50 was used as the statistical cutoff value after 5Aza-dC and/or TSA treatment to identify upregulated candidate genes.

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Project description:Analysis of 2 cultured normal lung cell lines, Normal Human Bronchial Epithelial (NHBE) and Human Small Airway Epithelial (SAEC) cells (Lonza, Walkersville, MD), following treatment with 5-aza-dC to induce DNA demethylation. These results provide insight into the role of epigenetic alterations, specifically demethylation, in differential gene expression in various lung neoplasms. Two normal lung cell lines, NHBE and SAEC, were treated with 5uM 5-aza deoxycytidine for 72 hours and Trichostatin A for 24 hours prior to harvesting total RNA for expression array analysis using the Affymetrix Human Genome U133 Plus 2.0 expression platform. Signal intensity and statistical significance was established for each transcript using dChip version 2005. Two-fold increase based on the 90% confidence interval of the result and expression minus baseline >50 was used as the statistical cutoff value after 5Aza-dC and/or TSA treatment to identify upregulated candidate genes.

Project description:To assess transcriptional regulation by DNA demethylation in SAEC, we carried out a microarray analysis of SAEC treated with a demethylating agent (5-aza-dC) and a HDAC inhibitor (TSA). We used the Agilent SurePrint G3 Human Gene Expression 8x60K v3 microarray which contains probes for 26,083 Entrez genes and 30,606 lncRNAs. Overall design: SAECs were treated with 3 μg/ml of 5-aza-dC on day1, 3 and 1 μg/ml of TSA on day4. Total RNA was extracted on day5, and microarray analysis was carried out using Agilent SurePrint G3 Human Gene Expression 8x60K v3.

Project description:To investigate the biochemical and genetic alterations that occur in response to cigarette smoke exposure among airway epithelial cells from different sites in the lungs, we performed microarray-based analysis using small airway epithelial cells (SAEC) and normal human bronchial epithelial cells (NHBE) following 24 h of cigarette smoke extract (CSE). In microarray-based analysis, the small airway showed higher susceptibility to CS compared to the large airway, such as enhanced expression of inflammatory-related pathways including the TNF signaling pathway. Among the TNF-related genes, PTGS2, also known as COX-2, showed the greatest difference in expression levels, with higher CSE-induced increments of both mRNA and protein expression in SAEC compared to NHBE. Overall design: SAEC and NHBE were subjected to 2.5% CSE for 24h. Microarray-based analysis was used to identify gene and protein expression profiles that are altered by CSE exposure in both cell types.

Project description:The goal of this study is to identify the gene expression changes caused by exposure of to the DNMT inhibitor 5-aza-2'-deoxycytidine (5Aza) and HDAC inhibitor Trichostatin A (TSA). We performed rRNA-depleted RNA sequencing of the untreated and drug-treated MCF7 breast cancer cell lines and carried out differential gene expression analysis. Although 5Aza caused a stranger demethylation effect than TSA, there were fewer differentially expressed gene in the 5Aza-treated MCF7 than the TSA-treated cells.

Project description:The goal of this study is to identify the DNA methylation changes caused by exposure of to the DNMT inhibitor 5-aza-2’-deoxycytidine (5Aza) and HDAC inhibitor Trichostatin A (TSA). We performed whole-genome bisulfite sequencing of the drug-treated MCF7 breast cancer cell lines and compare their DNA methylation profile with the untreated MCF7 (see E-MTAB-2014). While MCF7 treated with both drugs experienced global loss of DNA methylation, the 5Aza induced stronger demethylation than TSA.

Project description:Treatment-related DNA hypermethylation may play a role in creating drug resistant phenotypes by inactivating genes that are required for cytotoxicity, but there have been no genome-wide studies to systematically investigate methylation of individual genes following exposure to chemotherapy. We used microarrays and a pharmacologic unmasking protocol in isogenic cisplatin-sensitive and -resistant cell lines to identify genes that were down-regulated in cisplatin-resistant cells and could be re-activated by the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-dC). We identified several hundred genes that were down-regulated in each resistant cell line. Of these, 30 genes were common to > 2 cell lines, and/or reported to be down-regulated in previous studies. siRNA knockdown of two candidate genes increased cell viability with cisplatin treatment in sensitive parental cell lines Overall design: Cisplatin-sensitive and -resistant SCC cells and KB and KB cisplatin-resistant clones (n=2) were split to low density and treated with freshly prepared 5 microM 5-Aza-dC dissolved in 50% acetic acid/50% PBS or were mock treated with the same volume of vehicle in the media for 5 days. Subsequently, RNA was extracted and hybridized on Affymetrix U133A microarrays. Signal intensity and statistical significance was established for each transcript, and a 2-fold decrease in signal in each paired sensitive/resistant cell line in combination with 1.5-fold increase after 5Aza-dC treatment was used to identify candidate genes.

Project description:Treatment-related DNA hypermethylation may play a role in creating drug resistant phenotypes by inactivating genes that are required for cytotoxicity, but there have been no genome-wide studies to systematically investigate methylation of individual genes following exposure to chemotherapy. We used microarrays and a pharmacologic unmasking protocol in isogenic cisplatin-sensitive and -resistant cell lines to identify genes that were down-regulated in cisplatin-resistant cells and could be re-activated by the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-dC). We identified several hundred genes that were down-regulated in each resistant cell line. Of these, 30 genes were common to > 2 cell lines, and/or reported to be down-regulated in previous studies. siRNA knockdown of two candidate genes increased cell viability with cisplatin treatment in sensitive parental cell lines Cisplatin-sensitive and -resistant SCC cells and KB and KB cisplatin-resistant clones (n=2) were split to low density and treated with freshly prepared 5 microM 5-Aza-dC dissolved in 50% acetic acid/50% PBS or were mock treated with the same volume of vehicle in the media for 5 days. Subsequently, RNA was extracted and hybridized on Affymetrix U133A microarrays. Signal intensity and statistical significance was established for each transcript, and a 2-fold decrease in signal in each paired sensitive/resistant cell line in combination with 1.5-fold increase after 5Aza-dC treatment was used to identify candidate genes.

Project description:A unique feature of the tumour cells (Hodgkin/Reed-Sternberg (HRS)) of classical Hodgkin lymphoma (cHL) is the loss of their B-cell phenotype despite their B-cell origin. Several lines of evidence suggest that epigenomic events, especially promoter DNA-methylation, are involved in this silencing of many B-cell associated genes. Here we show that DNA-demethylation alone or in conjunction with histone-acetylation is not able to reconstitute the B-cell gene expression program in cultured HRS cells. Instead, combined DNA-demethylation and histone-acetylation of B cells induce a nearly complete extinction of their B-cell expression program and a tremendous up-regulation of numerous cHL characteristic genes including key players such as Id2 known to be involved in the suppression of the B-cell phenotype. Since the up-regulation of cHL characteristic genes and the extinction of the B-cell expression program occurred simultaneously, epigenetic changes may also be responsible for the malignant transformation of cHL. The epigenetic up-regulation of cHL characteristic genes thus play - in addition to promoter DNA-hypermethylation of B-cell associated genes – a pivotal role for the reprogramming of HRS cells and explain why DNA-demethylation alone is unable to reconstitute the B-cell expression program in HRS cells. Keywords: Epigenetic modification Overall design: 5-aza-dC treatment: The cHL derived cell lines L428, KMH2 and L1236 were treated with 5-aza-dC at a concentration of 5 µM for 5 days with drug and medium replacement after each 48 hours. Combined 5-aza-dC/TSA treatment: The Burkitt lymphoma derived cell lines (Daudi, Namalwa and Raji)were treated with 5-aza-dC at a concentration of 3 µM for 6 days. 5-aza-dC and medium was replaced at day 2 and 5. At the fifth day - in addition to 3 µM 5-aza-dC - cells were incubated for 24 hours with 625 nM TSA. The gene expression profiles of the untreated and treated cell lines were generated in duplicates.